17 September 2014

From fragment to lead: just remove (high energy) water

The proverb "well begun is half done" suggests that getting started comprises much of the work. Such is the case for fragments that bind to “hot spots,” sites on a protein that are particularly adept at binding small molecules and other proteins. Though fragment-to-lead efforts can give impressive improvements in potency, much of the binding energy of the final molecule resides in the initial fragment. In a new paper in ChemMedChem, Osamu Ichihara and colleagues at Schrödinger have asked why.

The researchers examined 23 published fragment-to-lead examples for which crystal structures and affinities of the fragment and lead were available and in which the fragment maintained its binding mode. They then used a computational tool called WaterMap to assess the water molecules displaced by both the initial fragment as well as the optimized molecule. They compared the calculated thermodynamic parameters (free energy, enthalpy, and entropy) of the water molecules displaced by the initial fragment (core hydration sites) or the bits added to it in the lead (auxiliary hydration sites).

When a protein is surrounded by water, water molecules bind just about everywhere. However, some of these water molecules may “prefer” to be in bulk solvent rather than, say, confined within a hydrophobic pocket on the protein. Perhaps not surprisingly, most of the water molecules displaced by ligands turned out to be of this “high-energy” or unstable variety. Also, the researchers consistently found that the core hydration sites were more unstable than the auxiliary hydration sites. In other words, fragments appear to displace the most unstable water molecules. Moreover, most of this higher energy was due to unfavorable entropy.

It is important to note that the focus here is on individual water molecules (or hydration sites) assessed computationally. The researchers are careful to stress that these may not correlate with thermodynamic parameters obtained by isothermal titration calorimetry (ITC). This is because ITC measures the entire system – protein, ligand, and all of the water – and factors such as protein flexibility can confound predictions.

The researchers summarize their findings as follows.
1) The presence of hydrogen bond motifs in a well-shaped small hydrophobic cavity is the typical feature of the hot spot surface  
2) Because of these unique surface features, the water molecules at hot spots are entropically destabilized to give high-energy hydration sites 
3) Fragments recognize hot spots by displacing these high-energy hydration sites
This provides a framework for understanding several phenomena. First, it describes the origin of hot spots. Second, it explains why much of the binding energy of an optimized molecule resides in the initial fragment; additional waters displaced are not as unstable as those displaced by the fragment, so they don’t give you as much bang for your atom. As a corollary, this might help explain the leveling off or decline in ligand efficiency often observed as molecules become larger.

The researchers go on to discuss specific examples of high-energy waters, noting that a water molecule involved in one or more hydrogen bonds may be particularly hard to replace because recapitulating the precise interaction(s) may be difficult. This is especially true for fragment-growing efforts (where one is likely to be limited in the choice of vector and distance) that aim to displace a high-energy water. Thus, the researchers suggest focusing on fragments that themselves displace high-energy waters, rather than trying to displace these later.

This seems like sound advice, but it likely reflects what folks already do. Since fragments that displace high-energy waters are likely to bind most effectively, won’t these be prioritized anyway? Regardless, this is an interesting and thought-provoking paper.


Dr. Teddy Z said...

So, this sits uneasy with me. I have said this before http://www.quantumtessera.com/your-computation-is-only-as-good-as-your-experimental-follow-up/
So, if someone used a completely different software package would they have identified the same waters and made the same conclusions?

Peter Kenny said...

It's worth remembering that hydrophobic association is non-local at the molecular level and that interactions between water molecules are cooperative and have a strong geometric dependence.

An alternative explanation of the decline in ligand efficiency with molecular size is that it's merely an artifact of the assumption that all measured IC50 and Kd values tend to 1 M in the limit of zero molecular size.